The goals of this project are to study mechanisms underlying the developmental and homeostatic regulation of the gene expression, posttranslation modification, and secretion of neuropeptides with a focus on the magnocellular oxytocin (OT) and vasopressin (VP) neurons in the hypothalamus. These neurons have historically served as valuable models for cell biological studies of peptidergic neurons, because of the relative ease by which the perikarya, axons, and nerve terminals of these cells in vivo can be isolated for anatomic, biochemical, and physiological analysis. Our general approach to experimental perturbation of molecular mechanisms in this system is to harness the regulatory elements in the OT and VP genes in transgenic mice in order to target specific molecules to these cells in vivo. The OT and VP peptide genes are good sources for regulatory controls since they are relatively abundantly and specifically expressed in these neurons in the CNS. The major problem has been to identify the critical regulatory elements in these genes which are responsible for the cell-specific expression. Recent transgenic studies have implicated various untranslated nucleotide sequence regions in the OT and VP genes which appear to contain these regulatory elements. Based on these studies, we have hypothesized that the 3.5 kb intergenic regions (between the OT and VP genes) in the mouse contains these cell-specific enhancers. In the past year, we have completely sequenced this intergenic region (IGR) and have found many putative regulatory motifs in this domain. In addition, two constructs containing the upstream regions of either the OT or the VP gene connected to the reporter protein, lacZ, followed by the IGR have been prepared for transgenic mouse experiments now in progress. Given success of our constructs in specific targeting in the OT and/or VP SV4O T-antigen) in an effort to generate OT and VP synthesizing cell lines. In the absence of such lines, we have developed several slice- explant systems to study OT and VP gene expression in vitro. These range from acutely prepared (<8 hr) slices to long-term cultured (3-4 weeks) slice-explants which remain "organotypic" in topography. The former (acute) systems have been very valuable for our studies on the role of calcium in immediate-early gene expression (e.g., c-fos), while the long- term cultures have been useful models for the study of OT and VP mRNA turnover and regulation in response to neurotransmitter and second message stimulation. In addition to the roller-culture versions of the "organotypic" cultures that we previously used, we have also recently employed "stationary" slice-explant cultures on millipore filters. The latter cultures also thin with time, remain even more "organotypic" structurally, and allow for the long-term survival of both OT and VP magnocellular neurons as well as the suprachiasmatic nucleus. Efforts to transfect cells in these slice-explants with various gene constructs using biolistic (particle mediated transfer of genes) techniques has met with partial success.